Quantitative detection of nitroxyl upon trapping with glutathione and labeling with a specific fluorogenic reagent
| Autor: | Gail M. Johnson, Tyler J. Chozinski, Alan D. Moghaddam, Katrina M. Miranda, Hsin Chih Chen, Debra J. Salmon |
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| Rok vydání: | 2012 |
| Předmět: |
Chromatography
Staining and Labeling Dimethyl sulfoxide Spectrum Analysis Inorganic chemistry Nitroxyl Glutathione Naphthalenes Biochemistry S-Nitrosoglutathione chemistry.chemical_compound Hydrazines chemistry Physiology (medical) Reagent TCEP Sodium azide Humans Nitric Oxide Donors Nitrogen Oxides Nitrite Amines Dimerization Fluorescent Dyes |
| Zdroj: | Free radical biologymedicine. 63 |
| ISSN: | 1873-4596 |
| Popis: | Donors of nitroxyl (HNO) have shown promise for treatment of stroke, heart failure, alcoholism and cancer. However, comparing the pharmacological capacities of various donors is difficult without first quantifying the amount of HNO released from each donor. Detection and quantitation of HNO has been complicated by the rapid self-consumption of HNO through irreversible dimerization, poor selectivity of trapping agents against other nitrogen oxides, and/or low sensitivity towards HNO. Here, an assay is described for the trapping of HNO by glutathione (GSH) followed by labeling of GSH with the fluorogenic agent, naphthalene-2,3-dicarboxaldehyde (NDA), and subsequent quantitation by fluorescence difference. The newly developed assay was used to validate the pH-dependence of HNO release from isopropylamine NONOate (IPA/NO), which is a dual donor of HNO and NO at physiological pH. Furthermore, varied assay conditions were utilized to suggest the ratios of the products of the reaction of GSH with HNO. At intracellular concentrations of GSH, the disulfide (GSSG) was the major product, but significant concentrations of glutathione sulfinamide (GS(O)NH₂) were also detected. This suggests that GS(O)NH₂, which is a selective biomarker of HNO, may be produced in concentrations that are amenable to in vivo analysis. |
| Databáze: | OpenAIRE |
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